Nucleic acids that regulate gene expression are widely considered to be potential therapeutics as well as important tools for gene function analysis. The potential of nucleic acid methods lies in their ability to regulate gene pathways by recognizing and binding complementary targets present in cells. However, the delivery of nucleic acids into mammalian cells remains a major challenge, as cells are naturally resistant to nucleic acid uptake. Additionally, they have a variety of mechanisms that degrade and destroy foreign nucleic acids both inside and outside the cell. Therefore, the creation of vectors that can non-toxically penetrate cellular membranes and deliver programmed nucleic acids without the aid of external transfection agents is necessary for the extension of these technologies to therapeutic application.
Polyvalent inorganic nanomaterials are now recognized as potential therapeutic agents in vivo, and in some cases are already FDA cleared for use as diagnostic tools [Rosi et al., Chem. Rev. 105 (4): 1547-1562 (2005); Taton et al., Science 289 (5485): 1757-1760 (2000)]. Because the surface of these particles can be associated with biomolecules through a variety of attachment strategies, they can be engineered through the choice of their surface ligands, to interact with well-known biological systems and pathways. For example, by modifying gold nanoparticles with a dense shell of duplexed siRNA, it possible to engage RNAi gene silencing in mammalian cells [Giljohann et al., J. Am. Chem. Soc. 131 (6): 2072-2073 (2009)]. These particles are particularly effective as RNAi gene regulation agents because they exhibit high cellular uptake without transfection agents [Rosi et al., Science 312 (5776): 1027-1030 (2006)], lack of acute toxicity, resistance to nuclease degradation [Seferos et al., Nano Lett. 9(1): 308-11 (2009)] and high stability in biological media. It is important to note that a growing body of work suggests that the gold nanoparticle-polynucleotide conjugate's ability to perform these various functions stems solely from the tightly packed arrangement of polynucleotides on the particles' surface [Giljohann et al., Nano Lett. 7 (12): 3818-21 (2007)]. In another example, a biomimetic synthetic high density lipoprotein (HDL) nanoconjugate can be constructed by modifying gold nanoparticles with a dense shell of phospholipids and APO1A, which is a biologically relevant protein [Thaxton et al., J. Am. Chem. Soc. 131 (4): 1384-5 (2009)]. HDL is a dynamic serum molecule protective against the development of atherosclerosis and resultant illnesses such as heart disease and stroke. Like biogenic HDL, this synthetic construct is capable of binding cholesterol in its hydrophobic phospholipid shell. Importantly, in both of these cases and many others, it is the dense polyvalent arrangement of biological ligands on the surface of inorganic nanoparticles that imparts their unique ability to interact with biological systems, regardless of their core material.
Although biological-inorganic nanomaterial hybrids possess desirable attributes, such as those used for diagnostics and therapeutics, concerns have arisen over the clearance/persistence and toxicity of the core material in vivo. Because these concerns are widely recognized as limitations for the use of nanomaterials in vivo, a universal approach is needed to create soft nanomaterials with tailorable surface functionalities that would maintain the properties of their inorganic nanoparticle bioconjugate counterparts. Attempts have been made to address these problems through a number of synthetic strategies, which includes micellar structures [Li et al., Nano Lett. 4 (6): 1055-1058 (2004); Liu et al., Chem-Eur J 16 (12): 3791-3797 (2010)].
Hollow nanoconjugates have attracted significant interest in recent years due to their unique chemical, physical, and biological properties, which suggest a wide range of applications in drug/gene delivery [Shu et al., Biomaterials 31: 6039 (2010); Kim et al., Angew. Chem. Int. Ed. 49: 4405 (2010); Kasuya et al., In Meth. Enzymol.; Nejat, D., Ed.; Academic Press: 2009; Vol. Volume 464, p 147], imaging [Sharma et al., Contrast Media Mol. Imaging 5: 59 (2010); Tan et al., J. Chem. Commun. 6240 (2009)], and catalysis [Choi et al., Chem. Phys. 120: 18 (2010)]. Accordingly, a variety of methods have been developed to synthesize these structures based upon emulsion polymerizations [Anton et al., J. Controlled Release 128: 185 (2008); Landfester et al., J. Polym. Sci. Part A: Polym. Chem. 48: 493 (2010); Li et al., J. Am. Chem. Soc. 132: 7823 (2010)], layer-by-layer processes [Kondo et al., J. Am. Chem. Soc. 132: 8236 (2010)], crosslinking of micelles [Turner et al., Nano Lett. 4: 683 (2004); Sugihara et al., Angew. Chem. Int. Ed. 49: 3500 (2010); Moughton et al., Soft Matter 5: 2361 (2009)], molecular or nanoparticle self-assembly [Kim et al., Angew. Chem. Int. Ed. 46: 3471 (2007); Kim et al., J. Am. Chem. Soc. 132(28): 9908-19 (2010)], and sacrificial template techniques [Réthoré et al., Small 6: 488 (2010)]. Among them, the templating method is particularly powerful in that it transfers the ability to control the size and shape of the template to the product, for which desired homogeneity and morphology can be otherwise difficult to achieve. In a typical templated synthesis, a sacrificial core is chosen, upon which preferred materials containing latent crosslinking moieties are coated. Following the stabilization of the coating through chemical crosslinking, the template is removed, leaving the desired hollow nanoparticle. This additional crosslinking step can be easily achieved for compositionally simple molecules, such as poly(acrylic acid) or chitosan [Cheng et al., J. Am. Chem. Soc. 128: 6808 (2006); Hu et al., Biomacromolecules 8: 1069 (2007)]. However, for systems containing sensitive and/or biologically functional structures, conventional crosslinking chemistries may not be sufficiently orthogonal to prevent the loss of their activity.